1. Field of the Invention
The present invention relates to a method for attaching a biological molecule, such as a recombinant protein derived from an antigen, an antibody or an enzyme, but also synthetic peptides and PNAs (peptide nucleic acids), to the surface of a support consisting of silica or metal oxides; it also relates to the supports thus prepared and to the uses which may be made of such a support.
2. Description of Related Art
Many methods exist for attaching biological molecules to silica supports. All of them involve a step of silanization of the silica which gives the surface the desired properties or functions. This functionalization then makes it possible to attach these compounds by adsorption, covalent coupling or complexation.
Firstly, there is the adsorption technique. Such a technique is, for example, described in patent applications EP-A-0,368,208 and WO-A-91/02980. In this case, the proteins can adsorb onto a support via several paths of interaction (1). The main ones are hydrophobic, polar and ionic interactions. These interactions depend on both the support, the protein and the medium under consideration.
The hydrophobic adsorption is carried out by a method for adsorption onto a flat silica support employed in the laboratory (2), which uses the interactions between a silanized surface with long alkyl chains and one or more hydrophobic regions of the protein.
However, in terms of orientation of the elements on the alkyl chains, the attachment is not at all uniform, and only a small portion of the elements will be really available for subsequent reactions, which may limit sensitivity.
Secondly there is the coupling technique. In this respect, covalent bonding methods also exist, which use silica functionalized with silanes which terminate with functions of type: amine, thiol or epoxy. These functions react on the chemically reactive groups of the accessible amino acids of the protein. The covalent bonding then takes place directly or via coupling agents.
The covalent bonding of the protein to a support is irreversible and generally takes place on several accessible functions of a protein. As a result of this, there is sometimes an inhibition of the activity of the molecule.
For example, patent application EP-A-0,874,242 relates to the silanization of a support (silica oxide) to which biological molecules can be attached in completely random manner. The attachment is carried out in several steps; firstly, activation of the silanols, by hydrolysis of the oxygen bridges; secondly binding between the silanols and an intermediate attachment molecule which can accept a ligand.
There are thus at least two steps, which lengthens the duration of reaction and is not always compatible with good results, biological or otherwise, which makes attaching the ligand more complex, and which does not make it possible to orient the ligand. The attachment to the intermediate attachment molecule is then random.
Patent application EP-A-0,272,792 has notably the same drawbacks as the application described above, since it proposes to attach antigens to particles, still in a random manner. Here again, there is a second intermediate step which consists in providing cyanogen bromide, which, via covalent bonds, will indirectly associate the particles and antigens.
So, these two patent applications EP-A-0,874,242 and EP-A-0,272,792 are thus very far removed from one of the main objects of the present invention. This object consists in orienting the biological molecules attached to the support.
However, it is possible to selectively attach, in an oriented manner, a protein which has a xe2x80x9ctagxe2x80x9d, for example lysine, as disclosed in the patent application FR 2 764 988, published Dec. 24, 1998.
All the same, there is no mention is this document of single-step attachment to a support consisting of silica or of metal oxide.
Thirdly, there is the chelation technique. In this way, a biological molecule can also be selectively attached to a support by chelation, according to a principle known to persons skilled in the art, such as the one employed for purifying proteins using the IMAC (Immobilized Metal ion-Affinity Chromatography) method on resins (3, 4). Such a protein possesses a histidine-rich labeling, termed xe2x80x9ctagxe2x80x9d. The difficulty of the method consists in generating a support having divalent ions which are required for the chelation. Such an approach is disclosed in the patent application FR 2 762 394, published Oct. 23, 1998. According to this invention, a method for attaching biological material makes it possible to optimize the complexation of this material with a metal complex, while onto the same decreasing, or even eliminating, any side reaction of adsorption of said material onto the metal complex. For this purpose, the method for attaching a biological material, of the invention, uses a coordination ligand compound, or a complex compound obtained from the latter, said ligand compound being in microparticulate form or in linear form, and consisting of at least one particulate or linear polymer, with an exposed hydrophilic surface and covalently-bonded free complexing groups.
The subject of the present invention is to enable the attachment of a biological molecule, such as a recombinant protein derived from an antigen, an antibody or an enzyme, but also of synthetic peptides and of PNAs (peptide nucleic acids) to silica or metal oxide supports. This immobilization uses interactions between the silanols of the silica, or the alcohol functions of the metal oxides, and a region which is specific to the protein. The specific properties of the biospecific support thus created are mainly a good orientation of the biological molecules or ligands at the surface and a support engendering very good signal to background noise ratios. In addition, this type of immobilization is simple and stable over time.
To this effect, the present invention relates to a method for attaching a biological molecule to the surface of a support consisting of silica or of metal oxide, the molecule comprising a specific binding site, characterized in that it consists in:
functionalizing the surface of the support by cleaning, using at least one solvent or an oxygen plasma or any other process for forming alcohol groups on the surface of the support, in order to render said surface hydrophilic
bringing the biological molecule directly into contact with said surface, and
functionalizing said support by attaching the specific binding site of the biological molecule to at least one of the alcohol groups borne by the surface of the support, which allows the molecule to be oriented for better reactivity.
According to one preferential embodiment, the method comprises, before the bringing into contact, a step of immersion of the functionalized surface in a sulfochromic mixture.
In all instances, the specific binding site of the biological molecule comprises the following characteristics:
it is an added amino acid sequence, i.e. added to the original sequence of the biological molecule,
this sequence is introduced into a preferred site of the original sequence, where it is exposed in a way which is relevant with respect to its function or to its properties, and
it contains in particular amino acids which are advantageous with respect to the desired function.
According to a first embodiment, the specific binding site of the biological molecule is a site rich in histidines and derivatives thereof, such as a site containing a sufficient density of histidines, in particular higher than or equal to 25%, and preferably higher than or equal to 33%.
More specifically, according to a second embodiment, the biological molecule and the specific binding site constitute a recombinant protein comprising at least two adjacent histidines called xe2x80x9ctagxe2x80x9d.
In particular, according to a third embodiment, the specific binding site of the biological molecule comprises at least four histidines, and preferably six adjacent histidines.
Whatever the case, the specific site is located within the recombinant protein or preferably at its N-terminal or C-terminal end.
The bringing into contact is carried out for 30 to 60 minutes.
More specifically, the bringing into contact is carried out for 45 minutes.
The immersion step is carried out for 5 to 30 minutes.
More specifically, the immersion step is carried out for 15 minutes.
The method is carried out at an optimum hydrogen potential (pH) as a function of the physicochemical properties of the biological molecule and of the specific binding site, preferably between 6 and 7.5.
The present invention also relates to a surface of a support, functionalized by the method described above.
In a preferential embodiment, the surface is flat.
In another embodiment, the surface consists of a bead.
The present invention also relates to the use of a functionalized surface of a support, as described above, or manufactured by the method described above, characterized in that it consists in carrying out assays to detect, in a liquid to be analyzed, the presence of any biological molecule capable of attaching, via a specific binding site, to the support.
In a preferential embodiment, a functionalized surface of a support, as described above, or manufactured by the method described above, is used for carrying out immunological assays to detect, in a liquid to be analyzed, the presence of antibodies specific for a natural protein, for example the P24 protein, by specific binding, in the same way as with the natural protein, to at least one recombinant protein, for example the RH24 or R24 protein.
This P24 protein is a recombinant protein of the type 1 human immunodeficiency virus (HIV-1) capsid.
The attached figures are given by way of indicating examples, and have no limiting nature with regard to the exact scope of the present invention.